The QIAseq FX Library Kit is a complete, all-enzymatic NGS library prep for high complexity DNA sequencing applications and is available in either 24-reaction or 96-reaction formats. This library prep is compatible with any Illumina sequencer and includes a 96-well plate containing either 24 or 96 dual-barcoded sequencing adapters (pierceable foil seal, allowing usage of defined parts of the plate). Library preparation from whole gDNA to sequencing-ready fragments takes just 2.5 hours and does not require any additional instrumentation or expensive glassware for DNA shearing – saving substantial time and cost. The QIAseq FX Library Kit is a fast, top-quality library prep for any whole genome or hybrid capture application designed with an easy-to-use protocol. Fragment sizes, input amounts and batch sizes are all customizable.

Buffers and reagents for DNA fragmentation (including end repair and A-addition), ligation and library amplification; for use with Illumina instruments; includes a plate containing 24 adapters with different barcodes (pierceable foil seal, allowing usage of defined parts of plate)

Buffers and reagents for DNA fragmentation (including end repair and A-addition), ligation and library amplification; for use with Illumina instruments; includes a plate containing 96 adapters with different barcodes (pierceable foil seal, allowing usage of defined parts of plate)

The QIAseq FX DNA Library Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

The QIAseq FX DNA Library Kit generates customizable, reproducible DNA fragmentation. A Fragmentation of samples using the QIAseq FX DNA Library Kit is highly customizable. Three separate libraries were produced to demonstrate the wide range of fragment sizes possible using the QIAseq FX kit. Libraries shown include inserts approximately 200 (green), 500 (blue) or 1000 (red) bp long. Data were generated using an Agilent® Bioanalyzer.

High Reproducibility

DNA samples with broad GC contents were fragmented using the QIAseq FX DNA Library it with either a 5- or 10-minute run time. Fragment sizes were highly reproducible for all samples for each run time.

Minimal GC bias compared to other enzymatic methods

Due to its highly sequence-independent fragmentation, QIAseq FX exhibits minimal GC bias, which is comparable to mechanical shearing. 100 ng genomic DNA was used as the input in comparative library preparation and sequencing. The QIAseq FX DNA Library Kit and the leading mechanical shearing method (Supplier C) produced similar and consistent coverage across a wide range of GC contents. An all-enzymatic protocol from Supplier N yielded much higher bias toward high % GC. 1 ng genomic DNA was used as the input in a similar experiment. Again, QIAseq FX and mechanical shearing provided similar and consistent coverage, outperforming other methods.

For this comparative distribution experiment, the input was 100 ng samples of an equimolar mixture of genomic DNA from three bacterial species with vastly different GC contents: Fusobacterium nucleatum with 27% GC; Escherichia coli with 50% GC; and Bordetella pertussis with 67% GC (with the exception of the sample undergoing tagmentation, as only specific input amounts are accepted). The single, narrow peak for the library created with QIAseq FX DNA Library Kit shows that the majority of genomic targets have very similar total coverage depth. This is comparable to DNA fragmented by mechanical shearing.

Superior coverage distribution and lower duplication rate

QIAseq FX technology with its minimal sequence bias ensures that the majority of genomic targets have very similar total coverage depth. This reduces the need for additional sequencing to bring low-coverage targets up to an interpretable coverage range, saving time and resources. Sequencing libraries generated by the QIAseq FX method have high complexity and low duplicate rates, overcoming the shortcoming commonly associated with other enzymatic fragmentation methods. (see figures: Superior coverage distribution and Lower duplication rate)

PCR-free libraries from as little as 100 ng input

Fully functional dual-barcoded adapters and optional high-fidelity library amplification reagents enable the use of QIAseq FX fragmentation and adapter ligation chemistries to generate PCR-free libraries in under two hours total workflow time. Without additional PCR library amplification, the QIAseq FX DNA Library Kit can consistently generate the higher than 2 nM PCR-free library concentration needed for sequencing on an Illumina MiSeq or NextSeq 500 – from as little as 100 ng input DNA. (see figure: No significant differences between coverage of high or low GC genomic regions)

Principle

QIAGEN’s QIAseq FX technology incorporates enzymatic DNA fragmentation into a streamlined, optimized protocol that combines fragmentation and adapter ligation in one reaction, saving time and preventing errors. The QIAseq FX kit contains a novel nuclease formulation that digests dsDNA in a random fashion without sequence preference, generating sequencing libraries with minimal bias. The fragment size is tunable simply by changing incubation time of the dsDNA with the nuclease, enabling different sequencing applications. Optimized enzyme and buffer compositions ensure high sequencing library yield. Streamlined library construction protocols also enable straightforward automation of library prep on different liquid-handling platforms.

The QIAGEN QIAseq FX DNA Library Kit provides a fast, fully enzymatic procedure, from DNA fragmentation to NGS library, with no cleanup steps until after adapters have been ligated to the sample DNA.

Procedure

Overview

The QIAseq FX DNA Library Kit consists of three, easy-to-follow steps starting from genomic DNA and ending with sequencer-ready NGS libraries. Beginning with combined DNA fragmentation and end modification, sample fragments are prepared allowing for the binding of dual-barcoded adapters. For samples that are less than 100 ng of input DNA or if large amounts of library DNA are required in later applications, an optional DNA amplification step can be performed with reagents conveniently provided in this kit.

DNA fragmentation and adapter ligation

Samples consisting of longer DNA fragments, such as genomic DNA or amplicons from long-range PCR, are first enzymatically sheared into smaller fragments. The median fragment size is dependent on the applications and sequencing read length, and can be adjusted by varying the DNA fragmentation reaction conditions. The fragmented DNA is directly end-repaired, and an 'A’ is added to the 3’ ends during the FX reaction ­– making the DNA fragments ready for adapter ligation. Following this step, platform-specific adapters are ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding dual-barcoded libraries to a flow cell for sequencing, allowing for PCR amplification of adapter-ligated fragments and for binding of standard Illumina sequencing primers.

DNA amplification

To ensure maximum yields from limited amounts of starting material, a high-fidelity amplification step can be performed using the reagents included in the QIAseq FX DNA Library Kit. The proprietary HiFi PCR Master Mix can evenly amplify DNA regions with vastly different GC contents, minimizing sequencing bias caused by PCR.

Library construction

Dual-barcoded, plate-format adapters are included with the QIAseq FX DNA Library Kit. Each well contains a single-use adapter consisting of a unique combination of two eight-nucleotide identification barcodes. By combining one D5 barcode and one D7 barcode in each ready-to-use adapter, QIAseq FX kits support up to 24-plex or 96-plex pooling prior to sequencing.

Cleanup and removal of adapter-dimers

Following library construction, the reaction cleanup and removal of adapter dimers can be achieved by using Agencourt® AMPure® XP Beads, enabling easy automation on various high throughput automation platforms.